High-Pressure Freezing and Freeze Substitution for Transmission Electron Microscopy

Worms were high pressure frozen in either a Bal-Tec HPM 010 (Bal-Tec AG, Liechenstein) or Leica HMP 100 (Leica Microsystems, Vienna) high pressure freezer and freeze substituted in 1% osmium tetroxide and 0.1% uranyl acetate in acetone over a period of 2 hours by the SQFS method of McDonald and Webb [45] (link). Infiltration of Epon epoxy resin was carried out by 15 minute incubations in 25, 50, and 75% acetone-resin mixtures on a rocker, then three 15 minute incubations in pure resin. Polymerization of resin was for 2 hours in a 100°C oven. Sections of 70 nm thickness were post-stained with 2% uranyl acetate in 70% methanol for 4 minutes and lead citrate (Reynolds, 1963) for 2 minutes. Images were viewed on a Tecnai 12 (FEI Inc., Hillsboro, OR, USA) transmission electron microscope operating at 120 kV, and images recorded with a Gatan Ultrascan 1000 CCD camera (Gatan Inc., Pleasanton, CA, USA). Some high magnification views of microtubule were taken out of focus in order to highlight protofilament patterns [48] (link), [49] (link).

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Hsu J.M., Chen C.H., Chen Y.C., McDonald K.L., Gurling M., Lee A., Garriga G, & Pan C.L. (2014). Genetic Analysis of a Novel Tubulin Mutation That Redirects Synaptic Vesicle Targeting and Causes Neurite Degeneration in C. elegans. PLoS Genetics, 10(11), e1004715.

Publication 2014

Tecnai 12 Ultrascan 1000 CCD camera

Acetone Citrate Epon Epoxy resin Freeze Methanol Microtubule Osmium tetroxide Polymerization Pressure Resin Transmission electron microscope Uranyl acetate Worms

Corresponding Organization :

Other organizations : National Taiwan University, University of California, Berkeley

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Variable analysis

independent variables

High pressure freezing method (Bal-Tec HPM 010 or Leica HMP 100)

dependent variables

Microtubule protofilament patterns

control variables

Freeze substitution in 1% osmium tetroxide and 0.1% uranyl acetate in acetone over 2 hours
Resin infiltration by 15 minute incubations in 25%, 50%, and 75% acetone-resin mixtures, followed by three 15 minute incubations in pure resin
Resin polymerization for 2 hours at 100°C
Section thickness of 70 nm
Post-staining with 2% uranyl acetate in 70% methanol for 4 minutes and lead citrate (Reynolds, 1963) for 2 minutes
Imaging using a Tecnai 12 transmission electron microscope operating at 120 kV, with images recorded using a Gatan Ultrascan 1000 CCD camera

controls

Positive control: None mentioned
Negative control: None mentioned

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